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- PDB-1nkh: Crystal structure of Lactose synthase complex with UDP and Manganese -

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Basic information

Entry
Database: PDB / ID: 1nkh
TitleCrystal structure of Lactose synthase complex with UDP and Manganese
Components
  • ALPHA-LACTALBUMIN
  • BETA-1,4-GALACTOSYLTRANSFERASE
KeywordsTRANSFERASE ACTIVATOR/TRANSFERASE / lactose synthease / UDP and MN binding / TRANSFERASE ACTIVATOR-TRANSFERASE COMPLEX
Function / homology
Function and homology information


Lactose synthesis / protein galactosylation / Keratan sulfate biosynthesis / Interaction With Cumulus Cells And The Zona Pellucida / Lactose synthesis / N-Glycan antennae elongation / lactose synthase / neolactotriaosylceramide beta-1,4-galactosyltransferase / beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase / N-acetyllactosamine synthase ...Lactose synthesis / protein galactosylation / Keratan sulfate biosynthesis / Interaction With Cumulus Cells And The Zona Pellucida / Lactose synthesis / N-Glycan antennae elongation / lactose synthase / neolactotriaosylceramide beta-1,4-galactosyltransferase / beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase / N-acetyllactosamine synthase / N-acetyllactosamine synthase activity / positive regulation of circulating fibrinogen levels / beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase activity / UDP-galactosyltransferase activity / regulation of acrosome reaction / penetration of zona pellucida / Golgi trans cisterna / lactose synthase activity / macrophage migration / lactose biosynthetic process / oligosaccharide biosynthetic process / development of secondary sexual characteristics / desmosome / acute inflammatory response / positive regulation of epithelial cell proliferation involved in wound healing / galactose metabolic process / binding of sperm to zona pellucida / angiogenesis involved in wound healing / protein N-linked glycosylation / Neutrophil degranulation / Transferases; Glycosyltransferases; Hexosyltransferases / Golgi cisterna membrane / beta-tubulin binding / epithelial cell development / alpha-tubulin binding / cytoskeletal protein binding / extracellular matrix organization / filopodium / epithelial cell proliferation / brush border membrane / lipid metabolic process / negative regulation of epithelial cell proliferation / lysozyme activity / manganese ion binding / basolateral plasma membrane / cell adhesion / positive regulation of apoptotic process / external side of plasma membrane / calcium ion binding / Golgi apparatus / protein-containing complex / extracellular space / extracellular region / identical protein binding
Similarity search - Function
Lactalbumin / Beta-1,4-galactosyltransferase / Galactosyltransferase, N-terminal / N-terminal region of glycosyl transferase group 7 / Galactosyltransferase, C-terminal / N-terminal domain of galactosyltransferase / Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain A / Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain A / Nucleotide-diphospho-sugar transferases / Lysozyme - #10 ...Lactalbumin / Beta-1,4-galactosyltransferase / Galactosyltransferase, N-terminal / N-terminal region of glycosyl transferase group 7 / Galactosyltransferase, C-terminal / N-terminal domain of galactosyltransferase / Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain A / Spore Coat Polysaccharide Biosynthesis Protein SpsA; Chain A / Nucleotide-diphospho-sugar transferases / Lysozyme - #10 / Glycoside hydrolase family 22 domain / Glycosyl hydrolases family 22 (GH22) domain signature. / Glycoside hydrolase, family 22 / C-type lysozyme/alpha-lactalbumin family / Glycosyl hydrolases family 22 (GH22) domain profile. / Alpha-lactalbumin / lysozyme C / Lysozyme / Lysozyme-like domain superfamily / Alpha-Beta Complex / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
: / URIDINE-5'-DIPHOSPHATE / Beta-1,4-galactosyltransferase 1 / Alpha-lactalbumin
Similarity search - Component
Biological speciesMus musculus (house mouse)
Bos taurus (cattle)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsRamakrishnan, B. / Qasba, P.K.
CitationJournal: J.Mol.Biol. / Year: 2001
Title: Crystal structure of lactose synthase reveals a large conformational change in its catalytic component, the beta-1,4-galactosyltransferase
Authors: Ramakrishnan, B. / Qasba, P.K.
History
DepositionJan 3, 2003Deposition site: RCSB / Processing site: RCSB
SupersessionJan 14, 2003ID: 1J8X
Revision 1.0Jan 14, 2003Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: ALPHA-LACTALBUMIN
B: BETA-1,4-GALACTOSYLTRANSFERASE
C: ALPHA-LACTALBUMIN
D: BETA-1,4-GALACTOSYLTRANSFERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)96,70519
Polymers93,7354
Non-polymers2,97015
Water10,953608
1
A: ALPHA-LACTALBUMIN
B: BETA-1,4-GALACTOSYLTRANSFERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,45010
Polymers46,8672
Non-polymers1,5828
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
C: ALPHA-LACTALBUMIN
D: BETA-1,4-GALACTOSYLTRANSFERASE
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,2559
Polymers46,8672
Non-polymers1,3887
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)55.469, 99.145, 102.261
Angle α, β, γ (deg.)90.00, 104.14, 90.00
Int Tables number4
Cell settingmonoclinic
Space group name H-MP1211

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Components

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Protein , 2 types, 4 molecules ACBD

#1: Protein ALPHA-LACTALBUMIN / / LACTALBUMIN / ALPHA


Mass: 14015.835 Da / Num. of mol.: 2 / Fragment: REGULATORY SUBUNIT OF LACTOSE SYNTHASE
Source method: isolated from a genetically manipulated source
Details: CHAINS A AND B FORM FIRST, C AND D SECOND LACTOSE SYNTHASE COMPLEX
Source: (gene. exp.) Mus musculus (house mouse) / Gene: alpha-lactalbumin / Plasmid: pET17.1 / Species (production host): Escherichia coli / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 / References: UniProt: P29752
#2: Protein BETA-1,4-GALACTOSYLTRANSFERASE / E.C.2.4.1.22, E.C.2.4.1.90, E.C.2.4.1.38 / B4GAL-T1 / BETA4GAL-T1 / BETA-1 / 4-GALTASE 1 / BETA-1 / 4-GALACTOSYLTRANSFERASE 1 / UDP-GALACTOSE: ...B4GAL-T1 / BETA4GAL-T1 / BETA-1 / 4-GALTASE 1 / BETA-1 / 4-GALACTOSYLTRANSFERASE 1 / UDP-GALACTOSE:BETA-N-ACETYLGLUCOSAMINE BETA-1 / 4-GALACTOSYLTRANSFERASE 1


Mass: 32851.617 Da / Num. of mol.: 2 / Fragment: CATALYTIC DOMAIN, RESIDUES 130-402
Source method: isolated from a genetically manipulated source
Details: CHAINS A AND B FORM FIRST, C AND D SECOND LACTOSE SYNTHASE COMPLEX
Source: (gene. exp.) Bos taurus (cattle)
Description: N-terminal carries a tag containing 13 amino acids
Gene: beta1,4-galactosyltransferase / Plasmid: pET23c / Species (production host): Escherichia coli / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21
References: UniProt: P08037, lactose synthase, N-acetyllactosamine synthase, beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase

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Non-polymers , 6 types, 623 molecules

#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#4: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#5: Chemical
ChemComp-PG4 / TETRAETHYLENE GLYCOL / Polyethylene glycol


Mass: 194.226 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C8H18O5 / Comment: precipitant*YM
#6: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
#7: Chemical
ChemComp-UDP / URIDINE-5'-DIPHOSPHATE / Uridine diphosphate


Type: RNA linking / Mass: 404.161 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C9H14N2O12P2 / Comment: UDP*YM
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 608 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.71 Å3/Da / Density % sol: 54.3 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 5.5
Details: PEG 4000, sodium citrate, pH 5.5, VAPOR DIFFUSION, HANGING DROP, temperature 298K
Crystal grow
*PLUS
pH: 5.6
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formulaDetails
120 mg/mlbeta4Gal-T11drop
210 mg/mlLA1drop
3100 mM1reservoirNaCl
4100 mMsodium citrate1reservoirpH5.6
512.5 %(w/v)PEG40001reservoir
617 mM1dropMnCl2

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X9B / Wavelength: 0.98 Å
DetectorType: ADSC QUANTUM 4 / Detector: CCD / Date: Aug 8, 2000 / Details: mirrors
RadiationMonochromator: graphite / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 2→25 Å / Num. obs: 57466 / % possible obs: 80 % / Observed criterion σ(I): 1 / Redundancy: 2.24 % / Biso Wilson estimate: 15.9 Å2 / Rsym value: 0.082 / Net I/σ(I): 15
Reflection shellResolution: 2→2.17 Å / Mean I/σ(I) obs: 1.1 / Rsym value: 0.75 / % possible all: 77
Reflection
*PLUS
Num. obs: 71688 / % possible obs: 99 % / Num. measured all: 224308 / Rmerge(I) obs: 0.082

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Processing

Software
NameVersionClassification
DENZOdata reduction
SCALEPACKdata scaling
AMoREphasing
CNS1refinement
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2→24.71 Å / Rfactor Rfree error: 0.003 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.251 5840 10.2 %RANDOM
Rwork0.196 ---
obs0.196 57466 79.5 %-
all-57466 --
Solvent computationSolvent model: FLAT MODEL / Bsol: 49.4612 Å2 / ksol: 0.306681 e/Å3
Displacement parametersBiso mean: 40.3 Å2
Baniso -1Baniso -2Baniso -3
1-11.93 Å20 Å24.21 Å2
2--1.25 Å20 Å2
3----13.18 Å2
Refine analyzeLuzzati coordinate error free: 0.33 Å / Luzzati sigma a free: 0.44 Å
Refinement stepCycle: LAST / Resolution: 2→24.71 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6396 0 179 608 7183
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.011
X-RAY DIFFRACTIONc_angle_deg1.6
X-RAY DIFFRACTIONc_dihedral_angle_d24.3
X-RAY DIFFRACTIONc_improper_angle_d1.01
X-RAY DIFFRACTIONc_mcbond_it1.411.5
X-RAY DIFFRACTIONc_mcangle_it2.292
X-RAY DIFFRACTIONc_scbond_it2.122
X-RAY DIFFRACTIONc_scangle_it3.042.5
LS refinement shellResolution: 2→2.13 Å / Rfactor Rfree error: 0.016 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.382 564 10.4 %
Rwork0.374 4878 -
obs--45.2 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2ION.PARAMION.TOP
X-RAY DIFFRACTION3WATER.PARAMWATER.TOP
X-RAY DIFFRACTION4UDP_PG.PARUDP_PG.TOP
Refinement
*PLUS
Highest resolution: 2 Å / Num. reflection obs: 54680 / Rfactor Rfree: 0.29 / Rfactor Rwork: 0.26
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.006
X-RAY DIFFRACTIONc_angle_deg1.5
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg24.3
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg1.01

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